Anti-CD3, CD16 CD56 IL-2 Human Activated Lymphocyte Treated with Anti-CD3, CD16, CD56 Monoclonal Antibody and IL-2 Seon-Min Hong 1, Dong-Wook Lee 1, Jin-Gu Kang 2, Han-Soo Kim 2, Sung-Hoon Cho 1 1 Biocell Co. Immune Research Institute and 2 Department of Medicine, Yonsei University College of Medicine ABSTRACT Background: Throughtout the last three decades, the therapy of leukemias and lymphoma has set the stage for curative cancer therapy in systemic malignant disease. This was the result of an integrated work of basic reaserch and clinical investigators leading to more aggressive albeit tolerable protocol of chemotherapy and radiotherapy. High dose therapy marks the most elaborated strategies in this field today. However, intensification of conventional therapeutic modalities as mentioned has to be based on new approaches and the exploration of new antineoplastic mechanisms. This insight has resulted in immune therapy of cancer. Among the cells of the immune system, natural killer () cells and T cells are of major interest for the development of therapeutic strategies. Methods: Cytotoxicity to target cells was measured by LDH release method, Characterization of activated lymphocyte was measured by Flow cytometry analysis. Anti-CD3, 16, 56 monoclonal antibody and IL-2 were used for the activation of and T cell. The analysis of effect of activated lymphocyte, in vivo, were used by Balb/c nude mouse. Results and Conclusion: Cytotoxicity to K562 cells was significantly higher in the mixture group of and T cells than that of a group of activating T cells. The survivors and the rate of reduction of size of tumor craft of nude mouse group treatment with activated lymphocyte was higher than that of the group without treatment with activated lymphocyte. Therefore, this results are suggested that the activated lymphocytes by anti-cd3, CD16 and CD56 can reduce the malignancy effect of lymphoma. (Immune Network 2005;5(1):11-15) Key Words: Activated lymphocyte, CD3, CD16, CD56, IL-2, natural killer cell, T cell, human, nude mouse, lymphoma, K562, raji Immune Network 11
12 Seon-Min Hong, et al. 된 정상세포도 죽이는 능력을 가진다. 그리고 형태적으 로는 큰 과립림프구이며, T세포의 표지인자나 표면단백 질인 Ig이 존재하지 않는다. 세포의 종양에 대한 세포 독성은 vitro상태에서 인터루킨-2 (IL-2)와 인터페론-α와 같은 세포조절물질이 존재하면 증가하게 된다. 세포 에서는 CD16 (FcγRIII)이 특이한 표면단백질이며 CD56 도 주된 표면 단백질이다. 그리고 T세포에서 발현되는 CD3는 결여되어있다(8). 암환자의 면역기능이 저하되면 면역결핍상태에서는 암 발생이 증가한다는 것이 알려지면서 암에 대한 면역 학적 치료법에 관하여 연구가 진행되어 왔다. 그 중 하나 가 종양숙주에 항암작용이 있는 면역세포를 투여하여 항암효과를 기대하는 양자면역요법이다(9). 1976년 인터 루킨-2의 발견으로 양자면역요법에 있어서 큰 발전을 이 루었다(10). 이 림프구 조절물질은 T세포뿐만 아니라 세포의 증식과 성장을 가능하게 하며 그들 세포에서 림프구 조절물질의 분비를 촉진, 증강시킨다(11). 본 연구에서는 현재 많이 연구되고 있는 세포면역치료 요법 중에서 세포와 T세포 그리고 T세포를 혼합 한 세포를 negative isolation 방법을 사용하여 분리하지 않고 공혈자로부터 채취한 혈액에서 림프구를 분리하고 anti-cd3, CD16, CD56 단일항체와 IL-2를 사용하여 활성 화시킨 림프구와 anti-cd3 단일항체와 IL-2를 사용하여 활성화시킨 림프구의 세포성상과 세포독성을 비교 분석 하여 활성화를 위해 사용된 항체에 따른 림프구의 활성 을 조사하고, Burkitt 림프종세포주인 사람의 B세포 림프 종을 유발하는 세포를 누드마우스에 주입한 후 종괴 를 유발시키고, anti-cd3, CD16과 CD56 단일항체에 의 해 자극되어 활성화된 사람의 림프구를 투여하여 종괴 에 대한 영향과 마우스의 생존도를 조사함으로써 림프 종에 대한 활성화 림프구의 영향을 알아보고자 한다. 재료 및 방법 세포. 마우스를 이용한 생체실험을 위해서 종괴를 형 성시키는 세포로는 사람의 B세포 림프종을 유발하는 Burkitt 림프종 세포주인 세포(ATCC: CCL-86)를 사 용한다. 그리고 세포독성은 사람의 leukemia세포주인 K562세포(연세대 소화기내과에서 배양중인 세포)를 사 용한다. 이 세포주들은 RPMI1640에서 10% 소태아 혈청 과 1%의 gentamycin을 첨가하고, 5% CO2 하의 습윤된 o 공기, 37 C에서 배양하였다. 실험동물. 6주령의 BALB/c 누드마우스를 구입하여, 청 정동물사역구역에서 동일 먹이와 동일 수분의 조건에서 사육하였다. 암세포의 이식. 세포 1 107개를 누드마우스의 오 른쪽 옆구리에 있는 피하조직에 주입하였다. 종양덩어 리는 늑골 융선을 덮으면서, 측면대부분을 차지하게 된 다. 이식 후 동일한 조건에서 사육하였다. 활성화림프구의 준비. 정상인 공혈자의 말초혈액을 헤 TM 파린 처리된 10 ml 진공채혈관(BD Vacutainer )에 채혈 하고 히스토파크(Histopaque, Sigma #1077-1, endotoxin tested, 밀도 1.077 g/ml) 밀도구배 원심법에 의해 림프구 를 분리한다. 분리된 림프구의 잔존 적혈구를 제거하기 위해서 0.83% 염화암모늄(NH4Cl)용액을 처리하고, 원심 apoptotic cells K-562 apoptotic cells Figure 1. Activated lymphocytes attacking, K562 cancer cell lines. Microscopic sections of cancer cells and activated lymphocytes incubated together at the rate of 1 10 for I hour ( 400, inverted microscope).
Human Activated Lymphocyte by Anti-CD3, CD16, CD56 Ab and IL-2 13 Table I. Cytotoxic effects according to characterization of activated lymphocytes. Cytotoxicity was analyzed by Elisa reader, using the method of LDH release. K562 cells and activated lymphocyte were incubated together at the rate of 1 10. (1), lymphocytes were activated by using anti-cd3 monoclonal antibody and IL-2. (2), lymphocytes were activated by using anti-cd3, CD16 and CD56 monoclonal antibody and IL-2. (3), lymphocytes were activated by using anti-cd3 and CD16 monoclonal antibody and IL-2. Cytotoxicity (%) 120 100 80 60 40 20 0 1 2 3 (%) 1 2 3 0.3±0.1 23.53±3.2 68.71±2.9 T 74±5.2 42.44±3.1 9.83±1.1 Table II. Cytotoxic effects to the target cells of and K562 cells. Cytotoxicity was analyzed by Elisa reader, using the method of LDH release,, K562 cells and activated lymphocyte were incubated together at the rate of 1 5 and 1 10, respectively. (1 and 2), the rate of K562 cells and activated lymphocytes are 1:5 and 1 10. (3 and 4), the rate of cellsa nd activated lymphocytes are 1 5 and 1 10, respectively. Cytotoxicity (%) 70 60 50 40 30 20 10 0 1 2 3 4 K562 1 1 5 3 1 5 2 1 10 4 1 10
14 Seon-Min Hong, et al. Table III. Tumor size (mm 3 ) and survival of mice observed at 3 days intervals in five nude mices after 7 days of tumor injection and subsequently injection of PBS, PBMC and activated lymphocytes, respectively day0 day3 day6 day9 day13 1 PBS 121 473.1 836.7 (6;2) 1306 (8;1) day 10, All dead 2 PBMC 130.9 367.6 913.9 (5;1) 1200.8 (8;2) day 10, All dead 3 M1 106.8 195.2 557.7 (8;1) 869.5 (10;2) day 13, All dead 4 M2 112.3 244.7 523.8 895.6 (11;2) day 13, All dead ( ), dead day and number of mice: (6;2), dead day was 6, and the number is 2 mice: M, cell and T cell. A C B D Figure 2. The photograph of tumor at 6 days in nude mice were injected PBS, PBMC and activated lymphocytes after 7 days of tumor injection. (A), Control, treatment with nothing after tumor injection, (B)(C)(D), a mouse treated with PBS, PBMC and activated lymphocytes, respectively.
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